Effects of membrane cholesterol manipulation on excitation-contraction coupling in skeletal muscle of the toad

Abstract

1. Single mechanically skinned fibres and intact bundles of fibres from the twitch region of the iliofibularis muscle of cane toads were used to investigate the effects of membrane cholesterol manipulation on excitation-contraction (E-C) coupling. The cholesterol content of membranes was manipulated with methyl-beta-cyclodextrin (MbetaCD). 2. In mechanically skinned fibres, depletion of membrane cholesterol with MbetaCD caused a dose- and time-dependent decrease in transverse tubular (t)-system depolarization-induced force responses (TSDIFRs). TSDIFRs were completely abolished within 2 min in the presence of 10 mM MbetaCD but were not affected after 2 min in the presence of a 10 mM MbetaCD-1 mM cholesterol complex. There was a very steep dependence between the change in TSDIFRs and the MbetaCD : cholesterol ratio at 10 mM MbetaCD, indicating that the inhibitory effect of MbetaCD was due to membrane cholesterol depletion and not to a pharmacological effect of the agent. Tetanic responses in bundles of intact fibres were abolished after 3-4 h in the presence of 10 mM MbetaCD. 3. The duration of TSDIFRs increased markedly soon (< 2 min) after application of 10 mM MbetaCD and 10 mM MbetaCD-cholesterol complexes, but the Ca(2+) activation properties of the contractile apparatus were minimally affected by 10 mM MbetaCD. The Ca(2+) handling abilities of the sarcoplasmic reticulum appeared to be modified after 10 min exposure to 10 mM MbetaCD. 4. Confocal laser scanning microscopy revealed that the integrity of the t-system was not compromised by either intra- or extracellular application of 10 mM MbetaCD and that a large [Ca(2+)] gradient was maintained across the t-system. 5. Membrane cholesterol depletion caused rapid depolarization of the polarized t-system as shown independently by spontaneous TSDIFRs induced by MbetaCD and by changes in the fluorescence intensity of an anionic potentiometric dye (DiBAC(4)(3)) in the presence of MbetaCD. This rapid depolarization of the t-system by cholesterol depletion was not prevented by blocking the Na(+) channels with TTX (10 microM) or the L-type Ca(2+) channels with Co(2+) (5 mM). 6. The results demonstrate that cholesterol is important for maintaining the functional integrity of the t-system and sarcoplasmic reticulum, probably by having specific effects on different membrane proteins that may be directly or indirectly involved in E-C coupling.

Figure 1. Effect of MβCD on E-C coupling in mechanically skinned fibres from toad muscle

Force responses induced by t-tubule depolarization in Na⁺-depolarizing solution (Depol) were almost abolished after a 2 min exposure to 10 mm MβCD in the presence of Na⁺-depolarizing solution. Note that MβCD was washed off the preparation in Na⁺-depolarizing solution before transfer to K⁺-repriming solution. Lowering [Mg²⁺] from 1 to 0.05 mm after exposure to MβCD could induce a large force response, indicating substantial Ca²⁺ release from the SR. Note that immediately following exposure to low [Mg²⁺] the preparation was briefly exposed to high-EGTA relaxing solution. The trace at the end of the panel represents the response to maximum Ca²⁺ activating solution (Max) at pH 7.10 and with 1 mm Mg²⁺. Artifacts on the force trace between solution changes are due to the transfer of the preparation through the air. Calibration bars: horizontal, 2 s during depolarization-induced and low-Mg²⁺-induced force responses and 30 s elsewhere; vertical, 50 μN. Fibre dimensions: L= 1.4 mm, D= 28 μm.

Figure 3. Effect of MβCD-cholesterol complexes on E-C coupling in mechanically skinned fibres from toad muscle

Force responses induced by t-tubule depolarization (Depol) were not significantly affected by 10 mm MβCD-1 mm cholesterol in the presence of Na⁺-depolarizing solution. Note that MβCD was washed out of the preparation in Na⁺-depolarizing solution before transfer to K⁺-repriming solution. Also, lowering [Mg²⁺] from 1 to 0.05 mm after exposure to MβCD-cholesterol induced a substantial Ca²⁺ release from the SR. Artifacts on the force trace between solution changes are due to the transfer of the preparation through the air. Calibration bars: horizontal, 2 s during depolarization-induced and low-Mg²⁺-induced force responses and 30 s elsewhere; vertical, 50 μN. Fibre dimensions: L= 1.5 mm, D= 48 μm.

Figure 8. Effect of MβCD-induced cholesterol depletion of the t-system on mechanically skinned fibres from toad muscle

A transient force response was elicited in K⁺-repriming solution in the presence of 10 mm MβCD about 12 s after transfer from Na⁺-depolarizing solution (A) or almost immediately following transfer from K⁺-repriming solution (B). C, after disruption of the sealed t-system with 50 μg ml⁻¹ saponin, no response could be elicited in the presence of MβCD. Lowering [Mg²⁺] from 1 to 0.05 mm after exposure to MβCD could still induce a substantial Ca²⁺ release from the SR. Note that immediately following exposure to low [Mg²⁺] the preparation was briefly exposed to high-EGTA relaxing solution. The trace at the end of each panel represents the response to maximum Ca²⁺ activating solution (Max). Artifacts on the force trace between solution changes are due to the transfer of the preparation through the air. Calibration bars: horizontal, 2 s during depolarization-induced, low-Mg²⁺-induced and saponin-induced force responses and 30 s elsewhere; vertical, 50 μN. Fibre dimensions: A, L= 1.5 mm, D= 43 μm; B, L= 2.5 mm, D= 43 μm; C, L= 1.9 mm, D= 38 μm.

Figure 6. The t-system of intact and mechanically skinned fibres maintains a [Ca²⁺] gradient and remains intact after exposure to MβCD but not saponin

A and B, confocal images of a toad skinned fibre before (A) and after (B) exposure to 10 mm MβCD for 10 min. C and D, confocal images of two different intact toad fibres loaded with fluo-3 that had been exposed to physiological solution for 1 h in the absence (C) or presence (D) of 30 mm MβCD prior to isolation from the iliofibularis muscle. Note the banded fluorescence pattern within the fibre, indicating that fluo-3 and Ca²⁺ remained trapped in the t-system in A-D. E and F, confocal images of a toad skinned fibre that had been loaded with fluo-3 before (E) and after (F) exposure to 100 μg ml⁻¹ saponin for 10 min. Note that the transverse fluorescence-banding pattern is no longer present in F, indicating that fluo-3 had been lost from the preparation. Scale bar: 10 μm.

Figure 7. The t-system membrane is depolarized by exposure to MβCD

Confocal laser scanning microscopy of a skinned fibre of toad that had been exposed to DiBAC₄(3) in the presence of Na⁺-HDTA solution (A), K⁺-HDTA solution (B) and 10 mm MβCD in the presence of K⁺-HDTA solution (C). Note that in B the preparation fluoresced intensely and the t-system was clearly evident, indicating that a membrane potential (more positive in the t-system lumen) was established in K⁺-HDTA solution. The ill-defined fluorescence pattern in C indicates that no significant membrane potential could be maintained in K⁺-HDTA solution when 10 mm MβCD was present. Scale bar: 10 μm.

Figure 2. Summary of the effect of MβCD on depolarization-induced force responses in toad skinned fibres

A, summary of results from 21 toad skinned fibres showing the decrease in the ability of preparations to respond to depolarization of the sealed t-system after exposure to 10 mm MβCD for different periods of time. B, summary of results obtained with 18 toad skinned fibres showing the decrease in the ability of preparations to respond to depolarization of the t-system after 2 min exposure to different concentrations of MβCD. The data were fitted by linear regression. The results in A and B were determined using the protocol described in Fig. 1 and are expressed as a percentage of the height of depolarization-induced force responses prior to exposure to MβCD or to an identical solution without MβCD (control). Values of n are given in parentheses. The continuous line in A is an exponential function of type y=Aexpbt, which best fitted the data points, where A= 97.3, b= −0.040 s⁻¹ and r = 0.90.

Figure 4. Summary of depolarization-induced force responses after 2 min exposure to 10 mm MβCD-cholesterol complexes in different ratios

The data points were best fitted to a Hill curve with variable slope of type: where n_H= 32.5, EC₅₀= 11.08, r = 0.90. The result is expressed as a percentage of the height of depolarization-induced force responses prior to 2 min exposure to each MβCD-cholesterol complex. Values of n are given in parentheses.

Figure 5. The effect of treatment with 10 mm MβCD on SR Ca²⁺ loading ability (relative response) and SR Ca²⁺ retention (Ca²⁺ retention index)

Summary of results showing the Ca²⁺ retention index (□; [Ca²⁺]_SR,90s/[Ca²⁺]_SR,30s; see Methods) and the Ca²⁺ loading ability (□; Relative response; see Methods) after 0, 2 and 10 min exposure to 10 mm MβCD. The data points for □ and □ were fitted to simple exponential and linear functions, respectively. Results are from 5 (□) and 9 (□) fibres. The data points at time 0 correspond to control responses either prior to exposure to 10 mm MβCD (□) or when the preparations were exposed to K⁺-HDTA solution without 10 mm MβCD for 10 min (□).

Figure 9. Summary of the effect of MβCD on tetanic force responses in bundles of intact muscle fibres

Tetanic force responses were abolished in the presence (□) but not in the absence (□) of 10 mm MβCD. Results are from 3 preparations in both cases. The result is expressed as a percentage of the height of the tetanic force response immediately prior to exposure to MβCD or just prior to the beginning of the control experiment. The continuous lines are exponential functions of types which best fitted the data points: and where A= 28.9 (□) and 341.8 (□), b= 0.012 min⁻¹, b₁= 0.016 min⁻¹, b₂= 0.022 min⁻¹; c= 72.6 and r = 0.71 (□) and 0.95 (□).